This application addresses broad Challenge Area (15) Translational Science, and specific Challenge Topic, 15-AI-106: Translational research focused on high priority pathogens and basic research focused on resistance mechanisms. Infections by Chlamydia trachomatis and C. pneumoniae cause diseases of the genital tract, eye, and respiratory tract in humans, and complications from these infections lead to chronic problems including trachoma, pelvic inflammatory disease, ectopic pregnancy, and perhaps other conditions. Millions of people worldwide are affected by these conditions, for which there are no effective vaccines. Discovery of useful vaccines and perhaps novel therapies against infection is complicated by the lack of a practical genetic system. The overall goals of this project work toward the development of a practical genetic system, through an examination of the mechanism of horizontal gene transfer by chlamydiae. Recent experimentation in our and other laboratories demonstrates that chlamydiae are actively recombinogenic in vitro and, very likely, in vivo. We have determined that these organisms are capable of mobilizing any of three antibiotic resistance genes between different strains and species, and stably retaining this DNA in the absence of selection. This includes measurable and frequent in vitro transfer of a tetracycline resistance marker to C. trachomatis from the pig pathogen C. suis. The mechanism of this transfer is not yet known. The following aims are proposed to utilize our developing technologies and accumulating preliminary data to elucidate the mechanism of natural genetic exchange in chlamydiae, and to exploit these findings to develop a practical genetic system for these organisms. In Aim 1 we will explore the mechanisms of recombination in Chlamydia spp., using differently resistant chlamydial strains already available in the laboratory. Aim 2 will use a recombination strategy as a tool for the mapping of map genes involved with known phenotypes in C. trachomatis. The final Aim will explore possible avenues for developing a practical technique for introducing genes into C. trachomatis. A common set of research tools will be used for each Aim, and these are largely already developed in our research group. Completion of these interactive Aims will significantly affect perceptions of how Chlamydia spp. interact in vivo, and may provide a practical transformation techniques that we can make available to the research community. They will also examine possible mechanisms that may allow C. trachomatis to acquire or transfer resistance genes in patient populations. PUBLIC HEALTH RELEVANCE: Millions of humans suffer serious diseases of the eye or genital tract caused by Chlamydia trachomatis. The study of possible vaccine candidates or logical therapeutic targets is limited by the lack of a practical genetic system. Our laboratories have developed recombination technologies that allow a study of the mechanisms involved in chlamydial recombination. Elucidating the mechanism of recombination in the chlamydiae will facilitate a rational approach to chlamydial transformation, which is also described in this proposal. Development of a workable genetic system will be important as researchers explore vaccine strategies to fight these bacteria.